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Secondary structure analysis of intramolecular DNA four‐way junction composed of phosphorothioate bonds
Author(s) -
Troisi Maria,
Parker Elliot,
Klein Mitchell,
Moorhead Gaston,
Alvarado Alice,
Herrada Hector,
Ghebreigziabher Daniel,
Shur Courtney,
Bell Anthony Jerome
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.471.23
Subject(s) - intramolecular force , dna , chemistry , circular dichroism , electrophoretic mobility shift assay , hmgb1 , biophysics , crystallography , stereochemistry , biology , biochemistry , gene , receptor , transcription factor
Research shows that cruciform and bent DNA can target the DNA‐binding cytokine, High Mobility Group B1 (HMGB1). HMGB1 was initially classified as a highly abundant architectural nuclear protein. It is now clear that HMGB1 also functions as a damage associated molecular pattern molecule (DAMP). And several studies link deleterious HMGB1 proinflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 garners a great deal of attention as a disease biomarker and therapeutic target. We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of 4WJs for ex vivo studies, the helical termini are modified to generate intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the intramolecular junction lattice to enhance nuclease resistance. In this study, the secondary structure of five intramolecular 4WJs are investigated vs . the DNA control, J1. J1 is an immobilized 4WJ that is composed of four asymmetric strands of DNA. Circular dichroism (CD) studies show that the secondary structure of each intramolecular 4WJ (DNA and PS constructs) is composed of B‐ and A‐form helices. Whereas, the DNA control J1 is devoid of A‐form helices. The melting temperature (T m ) of each intramolecular 4WJ is significantly higher than J1. The T m values for the intramolecular 4WJs are 25° – 30°C higher than J1. Electrophoretic mobility shift assays show that HMG proteins bind intramolecular 4WJs with similar affinity to J1. The data clearly shows that the lattice of an immobilized DNA 4WJ can be redesigned to enhance conformational stability and maintain: i) native secondary structure and ii) HMG binding affinity. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .